Iso-Electric Focusing

Figure 3.28 Iso-electric focusing. A protein which has an iso-electric pH of 6, in position A in the pH gradient is on the acid side of its iso-electric pH and will carry a positive (H+) charge. It will migrate electrophoretically towards the cathode. In position B, the charge on the protein will be negative and it will migrate towards the anode. In both cases, movement is towards its iso-electric pH position, where it will remain.

Figure 3.29 Iso-electric focusing of haemoglobin variants and derivatives. The...

Capillary procedures offer several advantages, including speed, resolution, sensitivity and technical simplicity, compared with the traditional methods on which they were based. An added advantage for iso-electric focusing in capillaries is the fact that it can be performed without a gel, but a coating on the internal surface of the capillary is usually required to reduce electroendosmosis. Similarly, isotachophoresis can be conveniently performed in capillary electrophoresis apparatus. 

In iso-electric focusing, an analyte will not move from a position where the buffer pH is the same as its iso-electric pH BECAUSE... 

Regardless of the rotor speed and maximum velocity, sedimentation (or flotation) will not occur in a solution of equal density to the sample. Iso-density methods use this lack of movement in a manner comparable to a pH gradient in iso-electric focusing techniques. The methods are a combination of sedimentation and flotation, achieved by using a density gradient that straddles the density of the particles concerned. On centrifugation, the particles sediment until they reach a solvent zone with the same density. This results in the development of a zone for each type of particle present in the sample. 

Iso-electric focusing techniques probably give the best resolution and many of the resulting bands are due to specific proteins. They are used mainly as a qualitative or a semi-quantitative technique due primarily to the large number of bands that develop and are of particular value when successive samples from the same source need to be compared for the presence or absence of a particular protein or for investigation of physical properties, e.g. p/. 

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Trieu-Cout, P. and Gripon, J. C. (1982). A study of proteolysis during Camembert cheese ripening using iso-electric focusing and two-dimensional electrophoresis. J. Dairy Res. 49, 501-510. 

Iso electric focusing (IEF) Determines the isoelectric point of the protein and detects modifications of the protein... 

Figure 12.4-1. An example of biosimilar epoetins. This figure shows epoetin alpha s marketed in Asia and South America analyzed by iso-electrical focusing. The different products show large differences in isotype content. Difference can also be seen between different batches of the same manufacturer (lA and IB IIA and IIB IIIA and IIIB). E is the epoetin alpha marketed in Europe.

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